Deubiquitinase USP13 regulates glycolytic reprogramming and progression in osteosarcoma by stabilizing METTL3/m6A/ATG5 axis

Reprogramming metabolism is a hallmark of cancer cells for rapid progression. However, the detailed functional role of deubiquitinating enzymes (DUBs) in tumor glycolytic reprogramming is still unknown and requires further investigation. USP13 was found to upregulate in osteosarcoma (OS) specimens and promote OS progression through regulating aerobic glycolysis. Interestingly, the m6A writer protein, METTL3, has been identified as a novel target of USP13. USP13 interacts with, deubiquitinates, and therefore stabilizes METTL3 at K488 by removing K48-linked ubiquitin chains. Since METTL3 is a well-known m6A writer and USP13 stabilizes METTL3, we further found that USP13 increased global m6A abundance in OS cells. The results of RNA sequencing and methylated RNA immunoprecipitation sequencing indicated METTL3 could bind to m6A-modified ATG5 mRNA, which is crucial for autophagosome formation, and inhibit ATG5 mRNA decay on an IGF2BP3 dependent manner, thereby promoting autophagy and the autophagy-associated malignancy of OS. Using a small-molecule inhibitor named Spautin-1 to pharmacologically inhibit USP13 induced METTL3 degradation and exhibited significant therapeutic efficacy both in vitro and in vivo. Collectively, our study results indicate that USP13 promotes glycolysis and tumor progression in OS by stabilizing METTL3, thereby stabilizing ATG5 mRNA and facilitating autophagy in OS. Our findings demonstrate the role of the USP13-METTL3-ATG5 cascade in OS progression and show that USP13 is a crucial DUB for the stabilization of METTL3 and a promising therapeutic target for treating OS.


Invasion assay, 3D tumor spheroid cell-invasion assay and chemotaxis assay
These functional experiments for assessing cell migration and invasion were conducted as previously described 1,2 .

Measurement of extracellular acidification rate (ECAR)
The ECAR was examined using a Seahorse XF96 Metabolic Flux Analyzer (Seahorse Biosciences, USA) according to the manufacturer's protocols. In brief, OS cells (3×10 4 ) in indicated groups were plated into each well of a Seahorse XF96 cell culture microplate. ECAR was evaluated by sequential adding glucose (10 mM), oligomycin (1 mM) and 2-deoxyglucose (2-DG, 80 mM).
Quantification of data was performed using XFe Wave software (Seahorse Biosciences) according to the manufacturer's instructions.

Measurement of glucose consumption, lactate production and ATP production
The culture medium of OS cells was obtained for glucose and lactate assays. Glucose levels were detected using a glucose assay kit (Nanjing jiancheng) and lactate levels were detected using a lactic acid assay kit (Nanjing jiancheng) according to the manufacturer's instructions. The level of intracellular ATP was detected using an ATP assay kit (Beyotime) according to the manufacturer's instructions.
Glucose metabolic flux analysis 13 C-Labeled intracellular metabolites were assessed as previously described 3 . In brief, 2×10 7 OS cells were incubated with 13 C 6 -labeled glucose (2g/L, Sigma) for 2h. Then, metabolites were collected, and those containing at least one 13 C atom were evaluated using an LC system equipped with a TripleTOF 5600 mass spectrometer (SCIEX, Framingham). Concentrations of 13 C 6 -labeled metabolites were normalized to cell number.

qRT-PCR
Total RNAs from OS cells and tissues were obtained using TRIzol reagent (TaKaRa Bio Inc., Japan). mRNA was reversely transcripted into cDNA using the PrimeScript RT Master Mix Kit

Autophagosome evaluation by GFP-mRFP-LC3 and transmission electron microscopy (TEM)
A GFP-mRFP-LC3 lentivirus (Obio, China) was used to evaluate autophagosome. The location as well as quantity of the autophagosomes were assessed using a confocal microscope (LSM710, Zeiss, Germany). Autophagic lysosomes were labeled red while autophagosomes were labeled red and green (yellow fluorescence). TEM was conducted as previously described 2 and images were acquired under an electron microscope (FEI, Hillsboro, OR, USA).

Immunohistochemistry (IHC)
IHC staining of 100 human OS specimens in tissue microarray (TMAs) were conducted using anti-USP13, -METTL3 and -ATG5 antibodies respectively. Samples were fixed in 4% paraformaldehyde followed by embedded in paraffin and was subsequently cut into 4-μm thick sections. Then IHC was performed by incubating sections with the indicated primary antibodies overnight and secondary antibody the next day.

Western blot
We used radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime) to lyse OS cells. After concentration examination, proteins were separated in SDS-PAGE, transferred onto PVDF membranes (Merck-Millipore), blocked with 5% BSA and incubated with specific primary antibodies at 4 ℃ overnight. Then, membranes were incubated with corresponding secondary antibodies at RT. Finally, the chemiluminescence reagent (Merck-Millipore) was applied to visualize the reacting bands.

Immunoprecipitation (IP)
A BCA protein assay kit (Beyotime) was used to detect the protein concentrations. Firstly, we used the protein A/G-agarose beads (Santa Cruz Biotechnology, USA) to preclear cell lysates for 1 h. Then the lysates were immunoprecipitated with the indicated antibodies at 4 ℃ overnight followed by incubation with protein A/G-agarose beads for 2 h at the next day. The immunocomplexes were then washed five times using RIPA lysis buffer and eluted by boiling for western blotting analysis.

In vivo ubiquitination assays
Endogenous METTL3 ubiquitination level was evaluated by immunoprecipitating cell lysates with anti-METTL3 antibody followed by immunoblotted with anti-Ub antibody. Exogeneous METTL3 ubiquitination level was assessed after co-transfecting Flag-tagged USP13 (WT or C345A mutant), Myc-tagged METTL3 and HA-tagged Ub in HEK 293T cells. Lysate proteins were immunoprecipitated and evaluated by immunoblotted with specific antibodies.

Measurement of m 6 A modification by LC-MS/MS
Quantification of RNA m 6 A modification was conducted by LC-MS/MS as previously described 4 . In brief, 200 ng mRNA and nuclease P1 were incubated in 20 μL buffer containing 25 mM NaCl, 2.5 mM ZnCl 2 for 2 h at 37 ℃ followed by addition of 2.2 μL NH 4 HCO 3 (1 M) and alkaline phosphatase, and incubated for 2 h at 37 ℃. After centrifugation at 13,000 rpm for 10 min at 4 ℃, 10 μL of the solution was further analyzed by LC-MS/MS.

RNA immunoprecipitation (RIP)
METTL3 RIP assay was performed with a Magna RIP TM RNA-Binding Protein Immunoprecipitation Kit (Millipore) according to the manufacturer's instructions. In brief, the cell pellet was lysed in RIP lysis buffer. Then 50 μL protein A/G magnetic beads were incubated with 5 μg anti-METTL3 antibody in RIP wash buffer at RT for 30 min. After that, lysates were immunoprecipitated with the beads-antibody complex in RIP immunoprecipitation buffer for 4 h at 4 ℃ before 10% RIP lysates were removed for input. IgG was used as a negative control. The magnetic bead-bound complex was centrifuged and washed at least five times, then the immunoprecipitated RNA was analyzed by qPCR.

RNA stability assay
To determine the mRNA stability of OS cells with the indicated treatments, actinomycin D (Sigma) at 5 μg/mL was added. After incubation for the indicated times, total RNA was isolated and the half-life of ATG5 was analyzed.